Renewable Energy 60 (2013) 127e136

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Renewable Energy
journal homepage: www.elsevier.com/locate/renene

Review

Relationships between cellulosic biomass particle size and enzymatic

hydrolysis sugar yield: Analysis of inconsistent reports in the
literature
Qi Zhang a, b, Pengfei Zhang b, *, Z.J. Pei b,1, Donghai Wang c
a
College of Mechanical Engineering, Yangzhou University, Yangzhou, Jiangsu 225127, China
b
Department of Industrial and Manufacturing Systems Engineering, Kansas State University, 2037 Durland Hall, Manhattan, KS 66506, USA
c
Department of Biological and Agricultural Engineering, Kansas State University, 150 Seaton Hall, Manhattan, KS 66506, USA

a r t i c l e i n f o a b s t r a c t

Article history: Cellulosic ethanol made from cellulosic biomass is a promising alternative to petroleum-based trans-
Received 5 September 2012 portation fuels. Enzymatic hydrolysis is a crucial step in cellulosic ethanol production. In order to better
Accepted 25 April 2013 understand the mechanisms of enzymatic hydrolysis, relationships between cellulosic biomass particle
Available online 29 May 2013
size and enzymatic hydrolysis sugar yield have been studied extensively. However, the literature contains
inconsistent reports. This paper presents an analysis of the inconsistent reports on the relationships in
Keywords:
the literature. It discusses the differences in the reported experiments from ve perspectives (biomass
Biofuel
category, particle size denition, sugar yield denition, biomass treatment procedure, and particle size
Cellulosic biomass
Enzymatic hydrolysis
level). It also proposes future research activities that can provide further understanding of the
Particle size relationships.
Sugar yield 2013 Elsevier Ltd. All rights reserved.

1. Introduction characteristics of cellulosic biomass have been identied as key

During the last three decades, consumption of petroleum-based
liquid transportation fuels (including gasoline, diesel, and jet fuels)
in the U.S. has increased by more than 30% [1]. These fuels account for
about 70% of total petroleum consumption in the U.S. [1]. In 2010,
more than 3 billion liters of petroleum were consumed in the U.S.
every day, and over 60% of them were imported [1]. Also, the price of
petroleum in the U.S. has almost doubled during the last ten years [1].
In addition, use of petroleum-based fuels contributes to accumula-
tion of greenhouse gas (GHG) in the atmosphere [2]. Therefore, it is
critically important to explore alternatives to petroleum-based liquid
transportation fuels [3e5]. One such alternative is cellulosic ethanol.
An essential step in cellulosic ethanol production is enzymatic
hydrolysis which converts cellulose into soluble sugars. A high
sugar yield in enzymatic hydrolysis is crucial to the cost-
effectiveness of cellulosic ethanol production. Several
factors affecting sugar yield in enzymatic hydrolysis, including
biomass particle size, degree of polymerization, crystallinity, and
accessible surface area [6e10]. In order to understand the mecha-
nisms of enzymatic hydrolysis and to increase sugar yield in
enzymatic hydrolysis, many studies have been done to investigate
the relationships between cellulosic biomass particle size and
enzymatic hydrolysis sugar yield. However, three different re-
lationships have been reported: negative (smaller particle size
produces higher sugar yield), neutral (particle size has no effects on
sugar yield), and positive (larger particle size produces higher sugar
yield).
This paper, for the rst time, presents an analysis of the incon-
sistent reports on the relationships. It discusses the differences in the
reported experiments from ve perspectives (biomass category,
particle size denition, sugar yield denition, biomass treatment
procedure, and particle size level). It also proposes future research
activities that can provide further understanding of the relationships.

Abbreviations: AFEX, Ammonia ber explosion; ASABE, American society of

agricultural and biological engineers; BET, Brunauer Emmet and Teller; MCC, 2. Two categories of cellulosic biomass
Microcrystalline cellulose; NREL, National renewable energy laboratory; SSA, Spe-
cic surface area; UV-A, Ultrasonic vibration-assisted. The cellulosic biomass used to investigate relationships be-
* Corresponding author. Tel.: 1 785 532 3729; fax: 1 785 532 3738.
E-mail addresses: qizhang@ksu.edu (Q. Zhang), pengfei@ksu.edu, xyz8106@
tween particle size and sugar yield can be classied into two cat-
gmail.com (P. Zhang), zpei@ksu.edu (Z.J. Pei), dwang@ksu.edu (D. Wang). egories: pure cellulose and lignocellulosic biomass. As summarized
1
Tel.: 1 785 532 3436; fax: 1 785 532 3738. in Fig. 1 and Table 1,when pure cellulose was used, negative

0960-1481/$ e see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.renene.2013.04.012
128 Q. Zhang et al. / Renewable Energy 60 (2013) 127e136
Negative
Relationship
Neutral
Positive
Pure cellulose Lignocellulosic biomass
Biomass category
Fig. 1. Reported relationships between particle size and sugar yield for two categories
of cellulosic biomass.
relationships were reported in a majority of the related studies.
When lignocellulosic biomass was used, three different relation-
ships were reported by different researchers.
Shewale and Sadana [15] reported a neutral relationship be-
tween particle size and sugar yield of pure cellulose MCC (micro-
crystalline cellulose). They used two particle sizes, 38 and 90 mm in
average. Their results are plotted in Fig. 2. It is noted that the re-
lationships between particle size and sugar yield were different for
different hydrolysis time. Sugar yield of smaller particles (38 mm)
was always higher except when hydrolysis time was 24 h. Because
they did not provide any information on variations in the sugar
yield data, it was not clear whether the differences were statisti-
cally signicant or not. Peters et al. [16] reported that sugar yield
was identical for three particle sizes (38e46, 46e63, and 74e
105 mm) of MCC. However, they obtained their different particle
sizes by screening a commercial brand of MCC (Avicel PH 102) with
screens of different mesh sizes. In most related studies, the different
particle sizes were obtained by milling followed by screening.
In the following, several observations will be discussed. Based
on these observations, hypotheses (or speculations) are formulated
to explain why different biomass categories might cause different
relationships between particle size and sugar yield. When appro-
priate, future research activities are also proposed to test these
hypotheses.
2.1. Ball milling had different effects on specic surface areas of
pure cellulose versus lignocellulosic biomass
One crucial step in enzymatic hydrolysis is the binding of
enzyme molecules to susceptible sites on cellulose surfaces [33e
35]. As specic surface area (SSA) of cellulosic biomass increases,
the number of enzymeecellulose bonds will increase, causing sugar
yield to increase [34,36e38]. Ball milling has been the most
Table 1
Reported relationships between particle size and sugar yield for two categories of
cellulosic biomass.
Biomass category Relationship Reference
Pure cellulose Negative [11e14]
Neutral [15,16]
Lignocellulosic biomass Negative [17e26]
Neutral [27]
Positive [28e32]
38 m 90 m
16
Surgar yield (mg/mL)
12
8
0
0 20 40 60
Hydrolysis time (hour)
Fig. 2. Relationships between particle size and sugar yield based on the data reported
by Shewale and Sadana [15].
commonly used method to reduce particle size for pure cellulose.
The extent to which particle size is reduced depends on milling
time. Longer milling time would produce smaller particle sizes. It
has been reported that, when ball milling was used to reduce
particle size of pure cellulose, SSA increased as particle size
decreased (or milling time increased). However, when ball milling
was used to reduce particle size of lignocellulosic biomass, SSA did
not always increase as particle size decreased (or milling time
increased).
For pure cellulose, Sinitsyn et al. [12] reported that SSA of cotton
linter doubled as particle size was reduced from 32 to 17 mm by ball
milling. Ouajai and Shanks [39] reported that SSA of MCC increased
by more than four times after 5-h ball milling.
For lignocellulosic biomass, Mikushina et al. [40] reported that
SSA of wood sawdust was increased by ve times after 5-h ball
milling. However, Gharpuray et al. [41] reported that there was no
difference in SSA of wheat straw after4-h versus 24-h ball milling. In
addition, even though the SSA of lignocellulosic biomass was
increased by milling, the SSA of cellulose (inside the lignocellulosic
biomass) might not increase as much due to the complex structure of
lignocellulosic biomass. In the literature, there are no investigations
about the SSA of cellulose inside lignocellulosic biomass for different
particle sizes.
Based on the above observations, it is hypothesized that, when
pure cellulose is used, ball milling can increase SSA (while reducing
particle size) and thereby increase sugar yield. When lignocellu-
losic biomass is used, ball milling does not change SSA (while
reducing particle size) and thereby does not increase sugar yield. It
is noted that other milling methods (such as knife milling and
hammer milling) have also been widely used for lignocellulosic
biomass. These milling methods will be discussed in Section 3.
2.1.1. Proposed future work
The hypothesis can be tested by investigating effects of ball
milling on SSA and sugar yield of pure cellulose and lignocellulosic
biomass. Various types of pure cellulose (such as cotton linter,
powdered cellulose, and MCC) and lignocellulosic biomass (such as
switchgrass, corn stover, and wheat straw) will be milled by ball
milling. For each biomass type, the milling process will be stopped
every 2 h (up to 48 h). Particle size, SSA, and sugar yield of milled
particles will be measured. Particle size will be determined ac-
cording to ASABE standard S424.1 [42]. SSA will be measured by
applying the BET equation to nitrogen adsorption data (this method
has been used by many researchers [33,43e45]). Sugar yield will be
 Q. Zhang et al. / Renewable Energy 60 (2013) 127e136
determined by following NREL laboratory analytical procedure [46].
At the end, for each biomass type, effects of ball milling on particle
size, SSA, and sugar yield will be obtained. The hypothesis will be
rejected if (1) smaller particle size for pure cellulose does not lead
to larger SSA and higher sugar yield, or (2) smaller particle size for
lignocellulosic biomass leads to larger SSA and higher sugar yield.
2.2. Before hydrolysis, pure cellulose particles of different size were
not pretreated but lignocellulosic biomass particles were pretreated
using different methods
In all reported studies using pure cellulose, no pretreatment was
applied to the cellulose particles of different sizes before enzymatic
hydrolysis. However, in the studies using lignocellulosic biomass,
particles of different sizes were pretreated (using various methods)
in some studies, but not pretreated in other studies. Such difference
in pretreatment might be a reason for the inconsistent results from
lignocellulosic biomass. More detailed discussion regarding pre-
treatment will be provided in Section 5.
2.3. Pure cellulose particles were smaller and in a narrower range
than lignocellulosic biomass
Fig. 3 shows the ranges of particle size in the reported studies
using pure cellulose and lignocellulosic biomass. The range of
particle size was from 0.78 to 100 mm for pure cellulose, and from
33 mm to 12 mm for lignocellulosic biomass. The larger range of
particle size might be a reason for the inconsistent results from
lignocellulosic biomass. More detailed discussion regarding particle
size range will be provided in Section 6.
3. Two denitions of particle size
Two denitions of particle size (sieve size versus screen size)
were used in reported experiments. As summarized in Fig. 4 and
Table 2,when sieve size was used, consistent (positive) relation-
ships were reported. When screen size was used, three different
relationships were reported.
Fig. 5 illustrates two denitions of particle size used in the
literature. For one denition, different sieve sizes were used in
milling to control particle size (one sieve size was used for one
particle size). The particle size was dened as the sieve size in
milling. For the other denition, only one sieve size was used in
certain types of milling methods (such as knife milling and hammer
milling) or no sieve was used in other types of milling methods
(such as ball milling). The particles produced from milling were
separated into different size ranges through a screening process
using a series of screens (each screen had a different mesh size). The
particle size was dened as the range between two neighboring
screen sizes.
cellulose
Pure
Lignocellulosic
biomass
2 4 6 8
Particle size (mm)
Fig. 3. The range of particle size for pure cellulose and lignocellulosic biomass.
129
Negative
Relationship
Neutral
Positive
Sieve size Screen size
Particle size definition
Fig. 4. Reported relationships between particle size and sugar yield for two denitions
of particle size.
In the following, several observations will be discussed. Based
on these observations, hypotheses (or speculations) are formulated
to explain why different particle size denitions might cause
different relationships between particle size and sugar yield. When
appropriate, future research activities are also proposed to test
these hypotheses.
3.1. Actual sizes of smaller sieve size particles were not signicantly
smaller than those of larger sieve size particles, but those of smaller
screen size particles were signicantly smaller than those of larger
screen size particles; the pores of smaller sieve size particles were
more damaged than those of larger sieve size particles, but those of
smaller screen size particles had similar damage as those of larger
screen size particles
3.1.1. Smaller sieve size particles might have similar external SSA
than larger sieve size particles while smaller screen size particles
might have larger external SSA than larger screen size particles
Mani et al. [47] measured size distribution of particles milled
using three different sieve sizes with four types of biomass (barley
straw, corn stover, switchgrass, and wheat straw). They calculated
the geometric mean length of the particles based on the ASABE
standard S424.1 [42]. Bitra et al. [48] measured size distribution of
particles milled using four different sieve sizes with three types of
biomass (corn stover, switchgrass, and wheat straw). Their results
with wheat straw are plotted in Figs. 6 and 7.The error bars in the
gures represent the standard deviation. Results with other types
of biomass are similar.
It can be seen from Figs. 6 and 7 that changes in geometric mean
length were much smaller than changes in sieve size. As sieve size
increased from 0.8 to 1.6 mm (Fig. 6), the average of geometric
mean length increased by only 0.06 mm which was much smaller
than the standard deviation (0.2 mm). As sieve size increased from
Table 2
Reported relationships between particle size and sugar yield for two denitions of
particle size.
Particle size denition Relationship Reference
10 12 Sieve size Positive [30e32]
Screen size Negative [17e26]
Neutral [27]
Positive [28,29]
130
Raw biomass
Milling with different sieve
sizes
Different particle sizes
(a) Sieve size
Fig. 5. Two denitions of particle size.
12.7 to 19 mm (Fig. 7), the average of geometric mean length
increased by only 1.7 mm, smaller than the standard deviation
(2.5 mm). This indicates that there was no signicant difference in
geometric mean length if difference in sieve size was less than
6 mm. Table 3 lists the sieve sizes used in those studies in which
positive relationships between particle size and sugar yield were
found. In all these studies, difference in sieve size was smaller than
4 mm. Therefore, it is likely that there was no signicant difference
in geometric mean length between the particles produced by
different sieves.
When particle size was dened as screen size, the average
particle length in one size range was usually signicantly different
from those in other size ranges. Zhu et al. [25] separated disk-
milled wood chips into four size ranges and hammer-milled
wood chips into two size ranges. They measured the average
particle length of each size range. Their results are shown in
Table 4. The difference in average particle length was comparable
to or even larger than the difference in the middle point of the size
range. Simonetis and Economides [49] reported a linear relation-
ship between the measured average particle length and average
screen size with a scope of 0.6. According to the results from these
two studies, it is likely that there was signicant difference in the
average particle length between the particles of different screen
sizes.
1.2
Geometric mean length (mm)
0.9
0.6
0.3
0 to 53e75 mm. Pedersen and Meyer [24] reported that the SSA of
0 1 2 3
Sieve size (mm)
Fig. 6. Geometric mean length of wheat straw particles milled using different sieve
sizes reported by Mani et al. [47].
Q. Zhang et al. / Renewable Energy 60 (2013) 127e136
20
Geometric mean length (mm)
Raw biomass
Milling with one sieve size or
15
without a sieve
10
Particles from milling
5
Screening with a series of
screens
0
0 20 40 60
Different particle sizes
Sieve size (mm)
(b) Screen size Fig. 7. Geometric mean length of wheat straw particles milled using different sieve
sizes reported by Bitra et al. [48].
3.1.2. Smaller sieve size particles might have smaller internal SSA
than larger sieve size particles while smaller screen size particles
might have similar internal SSA as larger screen size particles
For knife milling and hammer milling, it took longer milling
time to produce the same amount of particles when using smaller
sieve sizes. For example, in order to produce 1 kg of wheat straw
particles by knife milling, it took about 20 min when using a 0.25-
mm sieve size, but only 3 min when using an 8-mm sieve size [50].
Such difference in milling time for different sieve sizes could result
in different biomass internal SSA. It was reported by Gharpuray
et al. [41] that extended milling could damage the porous structure
of wheat straw and block the pores on biomass surface.
When screen size was used, particles of different screen sizes
were produced using one sieve size in milling. There was no sig-
nicant difference in milling time for different screen size particles.
Therefore, the degree to which the porous structure of cellulosic
biomass was damaged could be similar for different screen sizes.
The SSA of cellulosic biomass particles consists of external SSA
and internal SSA [33]. External SSA is determined by particle size.
Smaller particles have larger external SSA. Internal SSA is deter-
mined by the number and size of pores (and cracks) on biomass
surface. Particles would have larger internal SSA if there were more
and larger pores [51,52].
Based on the above observations, it is hypothesized that, when
sieve size denition is used, particles produced by a larger sieve size
might have approximately the same external SSA as those pro-
duced by a smaller sieve size (if the difference in sieve size is less
than a certain value, say 6 mm), but larger internal SSA, and
therefore larger total SSA; when screen size denition is used,
particles with a larger screen size might have approximately the
same internal SSA as those with a smaller screen size, but smaller
external SSA, and therefore smaller total SSA. Therefore, particles of
larger sieve sizes would produce higher sugar yields than those of
smaller sieve sizes, and particles of smaller screen sizes would
produce higher sugar yields than those of larger screen sizes.
Some studies seem to support this hypothesis. Zeng et al. [21]
reported that SSA of knife-milled corn stover particles increased
by ten times as particle size (screen size) decreased from 425e710
4 knife-milled wheat straw particles increased by up to 19 times as
particle size (screen size) decreased from 707e1000 to 53e149 mm.
It is noted that the results were obtained using one mill type, few
biomass types, and few particle size ranges. There are no studies
reporting effects of sieve size on total SSA of biomass particles.
 Q. Zhang et al. / Renewable Energy 60 (2013) 127e136 131

Table 3
Sieve sizes used in reported studies.

Sieve size (mm) Reference

1; 2 [30]
3.2; 6.5 [31]
1; 4 [32]

3.1.2.1. Proposed future work. This hypothesis can be tested by

investigating effects of sieve size and screen size on total SSA and
sugar yield of lignocellulosic biomass. The effects will be obtained
using knife milling and hammer milling and various biomass types
(such as switchgrass, corn stover, and wheat straw). For each
combination of mill type and biomass type, six sieves (with sieve
size of 0.25, 1, 2, 4, 6, and 8 mm respectively) will be used to pro-
duce particles. The particles milled using the 2-mm sieve will be
separated into ve size ranges (0.2e0.4, 0.4e0.6, 0.6e0.8, 0.8e1,
and 1e1.2 mm), and the particles milled using the 8-mm sieve will
be separated into another ve size ranges (1.4e1.7, 1.7e2, 2e2.4,
2.4e2.8, and 2.8e3.3 mm) using screens. Both total SSA and sugar
yield of the particles of each sieve size and each screen size (size Fig. 8. Effects of sieve size on cellulose content in milled poplar wood particles (after
range) will be measured. If particles of larger sieve sizes have Zhang et al. [32]).
signicantly larger total SSA and higher sugar yield than those of
smaller sieve sizes and particles of larger screen sizes have signif-
general, if the cellulosic biomass has higher cellulose and lower
icantly smaller total SSA and lower sugar yield than those of smaller
lignin content, its sugar yield will be higher.
screen sizes, the hypothesis will not be rejected.

3.2.1. Proposed future work

3.2. Compositions of different sieve size particles were different
while those of different screen size particles were the same
Zhang et al. [32] compared the cellulose content of poplar wood
particles milled using different sieve sizes. They found that the
particles of larger sieve size (4 mm) had higher cellulose content
than the particles of smaller sieve size (1 mm) for two milling
methods (knife milling and hammer milling), as shown in Fig. 8.
It has been reported that there is no signicant difference in
composition between particles of different screen sizes. Such re-
sults were reported by Chundawat et al. [19] with corn stover
particles of seven screen sizes, by Zeng et al. [21] with corn stover
particles of two screen sizes, and by Pedersen and Meyer [24] with
wheat straw particles of four screen sizes.
Based on observations discussed above, it is hypothesized that
milled particles of different sieve sizes might have different com-
positions. Particles of a larger sieve size might have higher cellulose
content or lower lignin content or both than those of a smaller sieve
size. However, milled particles of different screen sizes had the
same compositions. Sugar yield of cellulosic biomass is affected by
its composition especially the cellulose and lignin content. In
This hypothesis can be tested by investigating effects of sieve
size and screen size on composition and sugar yield of lignocellu-
losic biomass. The effects will be obtained using two mill types
(knife milling and hammer milling) and various biomass types
(such as switchgrass, corn stover, and wheat straw). The procedure
to prepare particles of different sieve sizes and screen sizes will be
similar to that described in Section 3.1. Compositions of different
particles will be determined by following NREL laboratory analyt-
ical procedure [46,53]. The hypothesis will be rejected if (1) there is
no signicant difference in composition (cellulose and lignin con-
tent) between particles of different sieve sizes, or (2) sieve size and
screen size have the same effects on composition and sugar yield.
3.3. Before hydrolysis, particles of different sieve sizes were pelleted
in most reported studies while those of different screen sizes were
not pelleted in most reported studies
In most studies where sieve size was used, particles from milling
were treated by pelleting and pretreatment before hydrolysis, as
shown in Fig. 9(a). In most studies where screen size was used,

Table 4
Particles from milling Particles from milling
Differences in average particle length measured by Zhu et al. [25].

Milling Size range Middle point Difference in Average Difference in

method (mm) of size rangea middle pointb particle average
Pelleting
(mm) (mm) length (mm) particle
lengthc (mm)

Disk 0.91e1.59 1.25 0.53 1.82 0.53

milling 0.53e0.91 0.72 0.33 1.29 0.57 Pretreatment Pretreatment
0.25e0.53 0.39 0.20 0.72 0.32
0.13e0.25 0.19 0.40
Hammer 0.64e1.27 0.96 0.48 1.92 1.39 Hydrolysis Hydrolysis
milling 0.32e0.64 0.48 0.53
a
Calculated as the average value of the lower and upper limit of the size range.
b
Calculated as the difference in middle point of two neighboring size ranges. (a) (b)
c
Calculated as the difference in average particle length of two neighboring size
ranges. Fig. 9. Two different treatment procedures for milled particles.
132 Q. Zhang et al. / Renewable Energy 60 (2013) 127e136

particles from milling were treated by pretreatment but not pel-

Negative
leting before hydrolysis, as shown in Fig. 9(b).
Two types of pelleting methods were used in the studies that
used particles of different sieve sizes. Ultrasonic vibration-assisted

Relationship
(UV-A) pelleting was used by Zhang et al. [30], and ring-die pel-

Neutral
leting was applied by Theerarattananoon et al. [31]. It was reported
that, with all other process variables being kept the same, particles
processed by UV-A pelleting produced higher sugar yield than

Positive
those not processed by UV-A pelleting [27], indicating that UV-A
pelleting itself had effects on sugar yield. Similar results were re-
ported by Theerarattananoon et al. [31] using ring-die pelleting. Glucose/pre-hydrolysis Glucose/pre-hydrolysis Glucose/pre-pretreatment
Consequently, effects of pelleting on sugar yield may interact with cellulose ratio biomass ratio cellulose ratio
effects of sieve size on sugar yield. Such interaction did not exist in Sugar yield definition
studies that used particles of different screen sizes.
Fig. 11. Reported relationships between particle size and sugar yield for different
Based on the above observations, it is hypothesized that effects
denitions of sugar yield.
of sieve size (or screen size) on sugar yield may be different
depending on whether or not biomass is pelleted before hydrolysis.
Table 5
3.3.1. Proposed future work Reported relationships between particle size and sugar yield for different denitions
This hypothesis can be tested by investigating effects of sieve of sugar yield.
size and screen size on sugar yield following both procedures in Sugar yield denition Relationship Reference
Fig. 9.The procedure to prepare particles of different sieve sizes and
Glucose/pre-hydrolysis cellulose ratio Negative [17e19,21e25]
screen sizes will be similar to that described in Section 3.1. If the
Positive [31]
same effects of sieve size (or screen size) on sugar yield are ob- Glucose/pre-hydrolysis biomass ratio Negative [20]
tained no matter whether pelleting is used or not, the hypothesis Neutral [27]
will be rejected. Positive [29,30,32]
Glucose/pre-pretreatment cellulose ratio Positive [18]

4. Different denitions of sugar yield

Three different denitions of sugar yield (glucose/pre-hydroly-
sis cellulose ratio, glucose/pre-hydrolysis biomass ratio, and
glucose/pre-pretreatment cellulose ratio) were used in reported
studies. Fig. 10 describes the sequence of four processes (size
reduction, pelleting, pretreatment, and hydrolysis) in cellulosic
ethanol production. The glucose/pre-hydrolysis cellulose ratio
represents the ratio (weight/weight) of glucose in post-hydrolysis
solution to cellulose in pre-hydrolysis biomass. The glucose/pre-
hydrolysis biomass ratio represents the ratio (weight/weight) of
glucose in post-hydrolysis solution to pre-hydrolysis biomass. The
glucose/pre-pretreatment cellulose ratio represents the ratio
(weight/weight) of glucose in post-hydrolysis solution to cellulose
in pre-pretreatment biomass. The three different ratios have the
same numerator (the weight of glucose in post-hydrolysis solution)
but different denominators.
As summarized in Fig. 11 and Table 5, when glucose/pre-
hydrolysis cellulose ratio was used, negative relationships were
reported by most researchers. When glucose/pre-hydrolysis
biomass ratio was used, three different relationships were re-
ported. When glucose/pre-pretreatment cellulose ratio was used,
positive relationships were reported.
In most reported studies, hydrolysis was performed using the
same amount of pretreated biomass by weight. When glucose/pre-
hydrolysis biomass ratio was used, a higher amount of glucose in
post-hydrolysis solution (Fig. 11) represented a higher sugar yield.
However, this is not necessarily true when glucose/pre-hydrolysis
cellulose ratio was used, because the cellulose content in pre-
hydrolysis biomass particles of different sizes could be different.
Ballesteros et al. [28] reported that, as particle size (screen size)
increased from 2e5 to 8e12 mm, the cellulose content of steam-
explosion-pretreated wood chips increased from 36 to 46%.
Therefore, when glucose/pre-hydrolysis cellulose ratio was used,
the higher amount of glucose in post-hydrolysis solution could be
offset by the larger cellulose content in pre-hydrolysis biomass. In
this context, it is possible that different relationships between
particle size and sugar yield could be obtained from the same test

Pelleting

Pre- Post-
Raw Liquid
Size reduction pretreatment Pretreatment pretreatment
biomass waste
biomass slurry

Post- Pre-hydrolysis
Solid
hydrolysis Hydrolysis
waste biomass
slurry

Post-
hydrolysis
solution

Fig. 10. Sequence of four processes in cellulosic biofuel manufacturing.

 Q. Zhang et al. / Renewable Energy 60 (2013) 127e136
by using pre-hydrolysis cellulose based or pre-hydrolysis biomass
based sugar yield denitions. For example, Ballesteros et al. [28]
reported that, after 8 min steam-explosion pretreatment at
210  C, the relationship was negative, because the sugar yield of
smaller particles (2e5 mm) was about 10% higher than that of
larger particles (5e8 mm) if sugar yield was dened as glucose/pre-
hydrolysis cellulose ratio. However, if sugar yield was dened as
glucose/pre-hydrolysis biomass ratio, the relationship became
positive, because the sugar yield of smaller particles was about 5%
lower than that of larger particles.
For some pretreatment methods (e.g., dilute acid pretreatment
and steam-explosion pretreatment), a part of cellulose in pre-
pretreatment biomass would be converted into glucose in pre-
treatment and considered as waste after pretreatment. Cellulose
recovery (a ratio of cellulose that remains in pre-hydrolysis biomass
to cellulose in pre-pretreatment biomass) measures cellulose loss
in pretreatment. A higher cellulose recovery means less cellulose
loss in pretreatment. Glucose/pre-pretreatment cellulose ratio
equals the product of glucose/pre-hydrolysis cellulose ratio and
cellulose recovery. Smaller particles usually generated higher
glucose/pre-hydrolysis cellulose ratios than larger particles. How-
ever, larger particles would usually lead to higher cellulose recov-
ery [18,23,28]. Therefore, it is possible that different relationships
between particle size and sugar yield could be obtained from the
same test if sugar yield was dened as glucose/pre-hydrolysis cel-
lulose ratio or glucose/pre-pretreatment cellulose ratio. For
example, Ballesteros et al. [18] reported that the glucose/pre-
hydrolysis cellulose ratio of smaller particles (2e5 mm) was
about 20% higher than that of larger particles (8e12 mm), but the
glucose/pre-pretreatment cellulose ratio of smaller particles was
0more than 20% lower than that of larger particles.
Based on the above observations, it is hypothesized that the
inconsistent relationships between particle size and sugar yield are
caused due to different sugar yield denitions used in different
studies.
4.1. Proposed future work
This hypothesis can be tested by comparing the relationships
between particle size and sugar yield with the same experimental
conditions but different sugar yield denitions. Various types of
lignocellulosic biomass will be milled using hammer milling. The
procedure to prepare particles of different sieve sizes and screen
sizes will be similar to that described in Section 3.1. Different par-
ticles will go through the same pretreatment (e.g., dilute acid
pretreatment) and enzymatic hydrolysis. Compositions of pre-
pretreated biomass and pre-hydrolysis biomass will be measured.
For each combination of biomass type and particle size, sugar yield
(using three denitions) will be obtained. If consistent relation-
ships between particle size and sugar yield are obtained based on
different sugar yield denitions, the hypothesis will be rejected.
5. Two procedures of treatment
Two procedures of treatment were used in reported studies e
one with pretreatment and the other without. As shown in Fig. 12
and Table 6, when pretreatment was not applied, consistent
negative relationships were reported. When pretreatment was
applied, three different relationships were reported. When biomass
was pretreated by ammonia ber explosion (AFEX) [19], hot water
[21], sodium carbonate [24], sodium chlorite [22], and steam ex-
plosion [18], negative relationships were reported. When biomass
was pretreated by lime [29], positive relationships were reported.
When biomass was pretreated by dilute acid [27,30e32], neutral or
positive relationships were reported.
133
5.1. Pretreatment could cause different changes in composition for
different particle sizes
Composition in pre-pretreated biomass was similar for different
particle sizes [19,21,25,31]. Pretreatment would remove hemi-
celluloses or lignin or both of them in order to increase accessibility
of cellulose [54e56]. When particles of different sizes were pre-
treated, lignin and hemicellulose may be removed with different
levels of success. Cullis et al. [57] used sodium hydrate pretreat-
ment to remove lignin from wood chips. They found that more
lignin was removed from larger chips (with the length of 5 mm)
than from smaller chips (with the length of 1.5 mm). Ballesteros
et al. [28] pretreated wood chips using steam explosion and sodium
chlorite. They reported that, after pretreatment, cellulose content
in larger particles (8e12 mm) was about 16% higher than that in
smaller particles (2e5 mm), whereas the lignin content in larger
particles was about half of that in smaller particles.
Based on the above observations, it is hypothesized that some
pretreatment methods (such as dilute acid, steam explosion, and
sodium hydrate) can cause different changes in composition for
different particle sizes. After pretreatment, larger particles have
lower lignin or hemicellulose content and higher cellulose content
than smaller particles. As a result, larger pretreated particles may
have higher (or at least similar) sugar yield than smaller pretreated
particles.
5.1.1. Proposed future work
This hypothesis can be tested by measuring the compositions of
pretreated lignocellulosic biomass particles of different particle
sizes. Various types of mills and biomass will be used. The proce-
dure to prepare particles of different sieve sizes and screen sizes
will be similar to that described in Section 3.1. Particles of different
sizes will go through the same pretreatment and enzymatic hy-
drolysis. Three pretreatment methods (dilute acid, steam explosion,
and sodium hydrate) will be tested. The hypothesis will be rejected
if (1) larger pretreated particles do not have statistically higher
cellulose content or lower lignin (or hemicellulose) content than
smaller pretreated particles, or (2) there is no signicant difference
in relationships between particle size and sugar yield with and
without pretreatment.
5.2. Pretreatment could cause different changes in specic surface
area for different particle sizes
It has been reported that, without pretreatment, particles of
smaller screen sizes might have larger SSA than those of larger
screen sizes. Zeng et al. [21] reported that SSA of smaller screen size
corn stover particles (53e75 mm) was about ten times higher than
that of larger screen size particles (425e710 mm). Pedersen and
Meyer [24] reported that SSA of smaller screen size wheat straw
particles (53e149 mm) was more than ten times higher than that of
larger screen size particles (707e1000 mm). SSA of lignocellulosic
biomass could be increased by many pretreatment methods [58].
Zeng et al. [21] observed that hot water pretreatment generated
many pores and hollows on surface of large particles but not on
surface of small particles. They believed that such pores and hol-
lows could increase surface area and make cellulose more exposed
and accessible to enzymes. Therefore, the difference in SSA be-
tween particles of different sizes would reduce signicantly after
pretreatment.
Based on the above observations, it is hypothesized that the
increase in SSA after pretreatment for larger particles might be
much greater than that for smaller particles. As a result, larger
pretreated particles may have higher (or at least similar) sugar yield
than smaller pretreated particles.
134 Q. Zhang et al. / Renewable Energy 60 (2013) 127e136
Negative
Relationship
Neutral
Positive
Without pretreatment With pretreatment
Treatment procedure
Fig. 12. Reported relationships between particle size and sugar yield for different
treatment procedures.
5.2.1. Proposed future work
This hypothesis can be tested by measuring SSA of pretreated
lignocellulosic biomass particles of different sizes. Different types
of biomass will be milled to particles with different sizes (similar to
the procedure in Section 3.1). These particles will go through the
same pretreatment and enzymatic hydrolysis. Three pretreatment
methods (dilute acid, steam explosion, and sodium hydrate) will be
tested. The hypothesis will be rejected if (1) the differences in SSA
between different particle sizes are not reduced signicantly after
pretreatment, or (2) there is no signicant difference in relation-
ships between particle size and sugar yield with and without
pretreatment.
5.3. Pretreatment could cause different changes in biomass
crystallinity for different particle sizes
Many researchers reported that lower biomass crystallinity led
to higher sugar yield [59e61]. Pretreatment could signicantly
change the difference in biomass crystallinity for different particle
sizes. For example, without pretreatment, the crystallinity of
miscanthus particles was 54% for particles with size of 250e
355 mm and 25% for particles with size of <63 mm [22]. After being
pretreated by sodium chlorite, the crystallinity of smaller
particles (<63 mm) increased remarkably (from 25 to 46%) but the
crystallinity of larger particles (250e355 mm) increased slightly
(from 54 to 56%). The difference in crystallinity between par-
ticles of different particle size was signicantly reduced after
pretreatment.
Based on the above observations, it is hypothesized that the
increase in biomass crystallinity after pretreatment for smaller
particles might be much greater than that for larger particles. As a
result, larger pretreated particles may have higher (or at least
similar) sugar yield than smaller pretreated particles.
Table 6
Reported relationships between particle size and sugar yield for different treatment
procedures.
Procedure Relationship Reference
Without pretreatment Negative [17,19e22,24,26]
With pretreatment Negative [18,19,21e24]
Neutral [27]
Positive [28e32]
5.3.1. Proposed future work
This hypothesis can be tested by measuring crystallinity of
pretreated lignocellulosic biomass particles of different sizes.
Different types of biomass will be milled to particles of different
particle sizes (similar to the procedure in Section 3.1). Crystallinity
will be measured using X-ray diffraction (this method has been
used by many researchers [33,59,62]). These particles will go
through the same pretreatment and enzymatic hydrolysis. Three
pretreatment methods (dilute acid, steam explosion, and sodium
hydrate) will be tested. The hypothesis will be rejected if (1) in-
crease in crystallinity for pretreated larger particles is not signi-
cantly greater than that for pretreated smaller particles, or (2) there
is no signicant difference in relationships between particle size
and sugar yield before and after pretreatment.
6. Different levels of particle size
Particle sizes used in most studies can be classied into two
levels: micron level (particle sizes were smaller than 1 mm) and
millimeter level (particle sizes were larger than 1 mm). As shown in
Fig. 13 and Table 7, when micron-level particle sizes were used,
negative relationships were reported by most studies. When
millimeter-level particle sizes were used, two different relation-
ships were reported.
6.1. Difference in specic surface area between large and small
particles at micron level was more signicant than that at
millimeter level
Particles of smaller screen size generally have larger SSA than
those of larger screen size. However, increase in SSA was not line-
arly related to decrease in screen size. Zhang et al. [63] reported
that the SSA of pan-milled cellulose powder increased by less than
0.2 m2/g as screen size decreased from 5 mm to 75 mm, whereas
increased by more than 0.5 m2/g as screen size decreased from 75
to 25 mm. Zhang et al. also observed that a slightly decrease in
screen size could result in a considerable increase in SSA for particle
sizes at micron level, but not so for particle sizes at millimeter level.
Therefore, when the particle size was at micron level, a consider-
able difference in SSA might exist for different particle sizes. In turn,
it could be expected that small particles would produce higher
sugar yield. However, when the particle size was at millimeter
level, the difference in SSA between different particle sizes would
Negative
Relationship
Neutral
Positive
Micron level Millimeter level
Particle size level
Fig. 13. Reported relationships between particle size and sugar yield for different
levels of particle size.
 Q. Zhang et al. / Renewable Energy 60 (2013) 127e136
Table 7
Reported relationships between particle size and sugar yield for different levels of
particle size.
Particle size level Effect Reference
Micron Negative [12e14,19e22,24e26]
Positive [29]
Millimeter Negative [18,23]
Positive [28,30e32]
be relatively small. In this context, particle size might not be a
dominant factor affecting sugar yield.
Based on the above observations, it is hypothesized that, when
particle size is at micron level, the differences in SSA and sugar yield
between different particle sizes are signicant. When particle size
is at millimeter level, the differences in SSA and sugar yield be-
tween different particle sizes are not signicant.
6.1.1. Proposed future work
This hypothesis can be tested by measuring SSA and sugar yield
of lignocellulosic biomass particles of different particle sizes.
Different types of biomass will be milled to particles of four
different size ranges e two smaller than 1 mm (0.2e0.4 and 0.4e
0.6 mm) and the other two larger than 1 mm (2e2.4 and 2.4e
2.8 mm). For each combination of biomass type and size range, SSA
of particles will be measured. These particles will go through the
same enzymatic hydrolysis and their sugar yield will be measured.
The hypothesis will be rejected if (1) the difference in SSA between
particles of 0.2e0.4 and of 0.4e0.6 mm are not signicantly larger
than that between particles of 2e2.4 and 2.4e2.8 mm, or (2) the
difference in sugar yield between particles of 0.2e0.4 and of 0.4e
0.6 mm are not signicantly larger than that between particles of
2e2.4 and 2.4e2.8 mm.
6.2. Effects of pretreatment on the relationships between particle
size and sugar yield for particle sizes at micron level were more
signicant than those for particle sizes at millimeter level
As discussed in Section 5, relationships between particle size
and sugar yield were affected by pretreatment. For particle sizes at
micron level, negative relationships were reported with and
without pretreatment. Pedersen and Meyer [24] reported that,
without pretreatment, sugar yield of smaller particles (53e149 mm)
of wheat straw was more than 3 times higher than that of larger
particles (707e1000 mm) after 12-h hydrolysis. When processed by
oxidation with Na2CO3, sugar yield of smaller particles was only
25% higher than that of larger particles. Yoshida et al. [22] inves-
tigated relationships between particle size and sugar yield for
miscanthus with and without applying sodium chloride pretreat-
ment. They reported that, without sodium chloride pretreatment,
sugar yield for smaller particles (<63 mm) was about 110% higher
than that for larger particles (250e355 mm) after 72-h enzymatic
hydrolysis. With pretreatment, sugar yield for smaller particles was
only about 20% higher than that for larger particles. Similar phe-
nomena were reported by Zeng et al. [21] with corn stover. Without
pretreatment, sugar yield of smaller particles (53e75 mm) was
almost double that of larger particles (425e710 mm) after 24-h
hydrolysis. After processed by hot water, sugar yield of smaller
particles was only 5% higher than that of larger particles.
For particle sizes at millimeter level, pretreatment could change
the relationship from negative to positive. Ballesteros et al. [28]
studied sugar yield of wood chip with different sizes (all larger
than 1 mm) and different steam-explosion pretreatment condi-
tions. They reported that, after pretreated at 190  C, the relation-
ship was negative. Small particles (2e5 mm) produced about 80%
135
higher sugar yield than large particles (8e12 mm). However, after
pretreated at 210  C, the relationship became positive. Large par-
ticles produced about 40% higher sugar yield than small particles.
Based on the above observations, it is hypothesized that, when
micro-level particle size is used, sugar yield is more dependent on
conditions of pretreatment rather than particle size. Different re-
lationships between particle size and sugar yield may be obtained
with and without pretreatment. Relationships between particle
size and sugar yield may also be different when different pre-
treatment conditions are used.
6.2.1. Proposed future work
This hypothesis can be tested by measuring sugar yield of large
particles with different pretreatment conditions. Different types of
biomass will be milled to two particle size ranges (2e2.4 and 4e
4.6 mm). For each combination of biomass type and size range,
some particles will go through enzymatic hydrolysis directly. Other
particles will go through dilute acid pretreatment with different
conditions (e.g., different temperature, different acid concentration,
and different pretreatment time) and enzymatic hydrolysis. If the
relationships between particle size and sugar yield are consistent
for all cases, the hypothesis will be rejected.
7. Concluding remarks
It is of both academic and practical importance to know the
relationships between cellulosic biomass particle size and enzy-
matic hydrolysis sugar yield. The literature contains inconsistent
reports on this relationship. Some say that smaller particles pro-
duce higher sugar yield, some say that larger particles produce
higher sugar yield, and some say that particle size does not affect
sugar yield.
It is desirable to understand why such inconsistence exists.
Looking at relevant publications from ve perspectives, several
hypotheses are proposed in this paper. Future work to test these
hypotheses should be benecial to gain insights and understanding
on the relationship.
Competing interests
The author(s) declare that they have no competing interests.
Acknowledgments
This study is supported nancially by NSF award CMMI-
0970112.
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